Hybridization Protection Assay (Hpa); Dual Kinetic Assay (Dka) - Procleix TIGRIS Operator's Manual

S 1
ECTION
The Enzyme Reagent contains Reverse Transcriptase and RNA Polymerase enzymes.
a) Reverse Transcriptase uses the promoter/primer to create a DNA copy of the target
RNA. If the target is DNA, the process skips to step d.
b) The RNAse H activities of the Reverse Transcriptase degrades the original target
RNA.
c) The second primer binds to the new DNA copy and Reverse Transcriptase creates
a double stranded DNA duplex.
d) RNA polymerase uses transcription to make multiple RNA copies of the DNA
template.
e) The process repeats until the reagent supply is exhausted.
4. The amplified RNA, called "amplicon," is ready to be hybridized with the labeled nucleic
acid probe in the Hybridization Protection Assay.

Hybridization Protection Assay (HPA)

The Hybridization Protection Assay (HPA) process hybridizes the amplicon to single-stranded
nucleic acid probes that are labeled with an Acridinium Ester (AE) molecule, and then it selects
and detects the hybridized probes.
1. The system moves the MTU into the HPA Incubator, where the HPA Pipettor dispenses
Probe Reagent into it.
The Probe Reagent contains AE labeled oligonucleotides. The sequence of the probes
specifically complements the target amplicon.
2. If amplicon is present, it hybridizes to the probes.
3. The HPA Pipettor dispenses Selection Reagent into the MTU, then moves the MTU to the
Transition Incubator.
4. During the Selection phase, the hybridized and unhybridized probes are differentiated by
inactivating the AE label on the unhybridized probes.

Dual Kinetic Assay (DKA)

During DKA (detection), the chemiluminescent signal produced by the hybridized probe is
measured in a luminometer and is reported as relative light units (RLUs). Inactive AE will not
emit light in the detection process.
1. The system dispenses Auto Detect 1 and Auto Detect 2 into the MTU.
2. Auto Detect 1 and 2 react with the AE molecule, emitting photons (particles of light).
3. A luminometer reads the light signal, if present.
Internal Control is added to each reaction tube as part of the wTCR. The Internal
Control in this reagent confirms that the specimen processing, amplification, and
detection steps have been performed properly. The Internal Control signal in each tube
is differentiated from the analyte signal by the different kinetics of the AE labels. The
Internal Control amplicon is detected using a probe with rapid emission of light (flasher
signal). Amplicon specific to the analyte is detected using probes with relatively slower
kinetics of light emission, termed a "glower" signal. The PROCLEIX
differentiate between Internal Control and combined analyte signals, but do not
discriminate between individual signals.
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Part # 902649EN Rev. A
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PROCLEIX TIGRIS System Operator's Manual
Volume I: Operating the Instrument
®
Assays